Metcalfe, Kyle S. and Murali, Ranjani and Mullin, Sean W. and Connon, Stephanie A. and Orphan, Victoria J. (2021) Experimentally-validated correlation analysis reveals new anaerobic methane oxidation partnerships with consortium-level heterogeneity in diazotrophy. ISME Journal, 15 (2). pp. 377-396. ISSN 1751-7362. PMCID PMC8027057. https://resolver.caltech.edu/CaltechAUTHORS:20200413-101943907
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Abstract
Archaeal anaerobic methanotrophs (“ANME”) and sulfate-reducing Deltaproteobacteria (“SRB”) form symbiotic multicellular consortia capable of anaerobic methane oxidation (AOM), and in so doing modulate methane flux from marine sediments. The specificity with which ANME associate with particular SRB partners in situ, however, is poorly understood. To characterize partnership specificity in ANME-SRB consortia, we applied the correlation inference technique SparCC to 310 16S rRNA amplicon libraries prepared from Costa Rica seep sediment samples, uncovering a strong positive correlation between ANME-2b and members of a clade of Deltaproteobacteria we termed SEEP-SRB1g. We confirmed this association by examining 16S rRNA diversity in individual ANME-SRB consortia sorted using flow cytometry and by imaging ANME-SRB consortia with fluorescence in situ hybridization (FISH) microscopy using newly-designed probes targeting the SEEP-SRB1g clade. Analysis of genome bins belonging to SEEP-SRB1g revealed the presence of a complete nifHDK operon required for diazotrophy, unusual in published genomes of ANME-associated SRB. Active expression of nifH in SEEP-SRB1g within ANME-2b—SEEP-SRB1g consortia was then demonstrated by microscopy using hybridization chain reaction (HCR-) FISH targeting nifH transcripts and diazotrophic activity was documented by FISH-nanoSIMS experiments. NanoSIMS analysis of ANME-2b—SEEP-SRB1g consortia incubated with a headspace containing CH₄ and ¹⁵N₂ revealed differences in cellular ¹⁵N-enrichment between the two partners that varied between individual consortia, with SEEP-SRB1g cells enriched in ¹⁵N relative to ANME-2b in one consortium and the opposite pattern observed in others, indicating both ANME-2b and SEEP-SRB1g are capable of nitrogen fixation, but with consortium-specific variation in whether the archaea or bacterial partner is the dominant diazotroph.
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| Additional Information: | © The Author(s) 2020. This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/. Received: 10 April 2020; Revised: 28 July 2020; Accepted: 21 August 2020. The authors acknowledge the ROC-HITS science party, R/V Atlantis crew, and HOV Alvin pilots from cruise AT37-13 for their assistance with sample collection and processing. We would like to thank H. Yu for assistance with sediment incubations, and S. Lim for performing IC measurements. We are grateful to Y. Guan for his assistance with the nanoSIMS analysis, R. Hatzenpichler for early BONCAT-FACS experiments, M. Aoki (National Institute of Technology, Wakayama College, Japan) for design of the FISH probe ANME-2a-828, and M. Schwarzkopf and Molecular Technologies for designing a set of HCR-FISH probes for nifH mRNA. We thank G. Chadwick and three anonymous reviewers for their comments on this work. Funding for this work was provided by the US Department of Energy’s Office of Science (DE-SC0020373), the SIMONS Foundation Life Sciences Collaboration on Principals of Microbial Ecosystems, the National Science Foundation BIO-OCE grant (#1634002), and a Gordon and Betty Moore Foundation Marine Microbiology Investigator grant (#3780); (all to VJO). A portion of this research was performed under the Facilities Integrating Collaborations for User Science (FICUS) initiative and used resources at the DOE Joint Genome Institute and the Environmental Molecular Sciences Laboratory, which are DOE Office of Science User Facilities. Both facilities are sponsored by the Office of Biological and Environmental Research and operated under Contract Nos. DE-AC02-05CH11231 (JGI) and DE-AC05-76RL01830 (EMSL). KSM was supported in part by a National Science Foundation Graduate Research Fellowship and a Schlanger Ocean Drilling Fellowship. VJO is a CIFAR Fellow in the Earth 4D: Subsurface Science and Exploration Program. These authors contributed equally: Kyle S. Metcalfe, Ranjani Murali. The authors declare that they have no conflict of interest. | ||||||||||||||||||||
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| Subject Keywords: | Metagenomics; Microbial ecology; Stable isotope analysis; Symbiosis; Water microbiology | ||||||||||||||||||||
| Issue or Number: | 2 | ||||||||||||||||||||
| PubMed Central ID: | PMC8027057 | ||||||||||||||||||||
| Record Number: | CaltechAUTHORS:20200413-101943907 | ||||||||||||||||||||
| Persistent URL: | https://resolver.caltech.edu/CaltechAUTHORS:20200413-101943907 | ||||||||||||||||||||
| Official Citation: | Metcalfe, K.S., Murali, R., Mullin, S.W. et al. Experimentally-validated correlation analysis reveals new anaerobic methane oxidation partnerships with consortium-level heterogeneity in diazotrophy. ISME J 15, 377–396 (2021). https://doi.org/10.1038/s41396-020-00757-1 | ||||||||||||||||||||
| Usage Policy: | No commercial reproduction, distribution, display or performance rights in this work are provided. | ||||||||||||||||||||
| ID Code: | 102503 | ||||||||||||||||||||
| Collection: | CaltechAUTHORS | ||||||||||||||||||||
| Deposited By: | Tony Diaz | ||||||||||||||||||||
| Deposited On: | 13 Apr 2020 17:36 | ||||||||||||||||||||
| Last Modified: | 09 Feb 2022 20:37 |
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